Endo-β-1,4-D-mannanase (β-mannanase; EC 3.2.1.78) catalyses the random hydrolysis of manno-glycosidic bonds in mannan-based polysaccharides. Most β-mannanases degrade oligosaccharides down to DP4 (Biely and Tenkanen (1998) Enzymology of hemicellulose degradation, pages 25-47. In Harman and Kubiceck (ed) Trichoderma and Gliocladium, vol. 2, Taylor and Francis Ltd. London), however, residual activity has been demonstrated on mannotriose, indicating at least four subsites for mannose binding on the protein. The main end products of hydrolysis are often mannobiose and mannotriose, although significant amounts of mannose are also produced. Some β-mannanases are able to degrade crystalline mannan. In addition to hydrolysis, several β-mannanases including β-mannanase from Trichoderma reesei, have been shown to form transglycosylation products with either mannose or mannobiose as glycosidic bond acceptor.
β-mannanases have been isolated from a wide range of organisms including bacteria, fungi, plants and animals. Although mostly extracellular, some β-mannanases appear to be cell-associated. Their expression is often induced by growth on mannan or galactomannan, however, β-mannanase from T. reesei can also be induced by cellulose, while its expression is suppressed by glucose and other monosaccharides. Frequently multiple mannanases with different isoelectric points are found in the same organism, representing products from different genes or different products from the same gene, respectively.
In general, β-mannanases have moderate temperature optima between 40° C. and 70° C., except some β-mannanases from thermophiles (Politz et al. (2000) A highly thermostable endo-1,4-β-mannanase from the marine bacterium Rhodothermus marinus; Appl. Microbiol. Biotechnol. 53:715-721). The pH-optimum is in the neutral or acidic region, e.g. pH 5.0 for β-mannanase from T. reesei (Arisan-Atac et al. (1993) Purification and characterisation of a β-mannanase of Trichoderma reesei C-30; Appl. Microbiol. Biotechnol. 39:58-62). The molecular weights of the enzymes range between 30 kD and 80 kD.
WO 002008009673 discloses variants of Trichoderma reesei mannanases improved in thermal stability and low pH/pepsin resistance for the use in hydrolysis of galactomannan containing plant material, e.g. palm kernel expeller (PKE) and for the use in animal feed. For example, thermostability is required for feed additives that are incorporated in the feed mixtures prior to a pelleting procedure that comprises high temperatures. Additionally, mannanases applicable as feed additives need to be low-pH- and pepsin-stable and have to be active at low pH in order to be able to work efficiently in the stomach of e.g. monogastric animals.
However, the trend in the feed industry is to increase the pelleting temperatures further and further. Currently enzyme stability around 90° C. to 95° C. pelleting temperature is targeted to enable the use of enzymes throughout all industrially relevant feed production plants. Therefore the availability of a mannanase with improved thermal stability would be highly advantageous, as it would allow using the enzyme also in plants with high operation temperature.